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- Improvement of silver impregnation technique (protargol) to obtain morphological features of protists ciliates, flagellates and opalinates

The silver impregnation technique (protargol) offers for optical microscopy the best results in the revelation of morphologic characteristics of the structures of the cortex and endoplasma, as well as the form and disposition of the nuclei of protista ciliates, flagellated and opalinates.

The silver proteinate, used in the technique, acts as an impregnanting agent, on the level of the kinetossomes or basal corpuscles of the cilia and flagellum. With the impregnation of these it is placed in evidence the distribution of the somatic kineties and organization of the cilia and flagellum of the oral area of the protozoan, facilitating the counting and description of its components.

The modifications and improvements of the protargol technique started with the results presented by Bodian (1936, 1937), who used the technique for the revelation of cells of the nervous tissue.

The modifications of the technique described by several researchers (Dieckerman, 1995; Dragesco, 1962; Foissner, 1991; Hiller, 1991; McCoy, 1974; Montagnes & Lynn, 1987; Moskowitz, 1950; Ng & Nielsen, 1977; Repak & Cribbins, 1966; Tuffrau, 1964, 1967; Wilbert, 1975; Zagon, 1970), were made for optimal results and adapted to the material available in their laboratories, thanks to a vast possibility of variation of the technique.

Tuffrau (1964, 1967) suggest that a minimum of albumin-glycerol of Mayer solution should be used to attach the cells in the slide, diluted in a proportion of 1:10, to form a very fine layer on the slide. The addition of the albumin-glycerol of Mayer on the cells can be done directly on the slide or in a small glass recipient or clock glass; in this case, after homogenization, the cells are distributed diluted in Mayer solution over several slides or coverslips. It is adequate the attachment of the cells in coverslips, to facilitate posterior observation under the microscope of both faces of the impregnated specimen.To avoid the detachment of the cells during the processing of the technique, Tuffrau (1967) suggest the immersion of the slides in the alcool-formol v/v, for 3 minutes. Foissner (1991) suggest the use of the albumin¾glycerol after a month of stability in the refrigerator. For the attachment of the cells on the slides or coverslips is adequate as an alternative, the use of polylysine. Which was not used before in the impregnation technique.

The time of impregnation of the cells may vary, as well as the temperature of the incubator.

The concentration of the hydroquinone can be from 0.4% to 1%. It is adequate a lower concentration. The preparation should be revealed first at very short times and after, the tonality of the impregnation controlled under the microscope. The recommended tonality resembles the color of Whisky. Tuffrau suggests a imersion in 5% sodium thiosulfate, if the specimens are too dark.

The fixation of the impregnation in sodium thiosulfate should be made between 30 seconds and one minute. Because this may darken the preparation, this stage is very important for the preservation of the impregnated cells for long term storage of the slides in collections.

Finally is adequate the use of the Entellan in stead of balsam from Canada for the mounting of the slides.

In addition to the technique of the protargol, other impregnation techniques by silver can be used, such as the technique of Chatton & Lwoff (1930), of Klein (1915) and of Fernando-Galeano (1976), that also offer great results in the revelation of important morphological data. But the first, which reveals most of the structures of the cortex of the cell very well, does not evidence the organelles and nuclei of endoplasma. Klein's technique, also called dry silver, does not reveal the whole structures of the cortex, but it evidences the plates of the submembranal cytoskeleton, that exist in several ciliates species. These structures are not very proeminent with protargol, that complements the study of certain protista species. The technique of Fernando-Galeano, also called the technique of the carbonate of silver amoniacal, offers great results like protargol, but it does not allow the preparation of definitive slides for storage in collections. It is necessary to underline that the results of the technique of the protargol are complements of the observation of the protozoan in vivo.


Acknowledgments — I would like to thank the trainees of the laboratory (Anderson Bruno de Matos, Flávio Joppert, Fábio Lima Custódio and Maurício Guimarães Gomes Moreira) and to Prof. Ulisses Lins of the Section of Electronic Microscopia of Inst. of Microbiology-UFRJ, for the access to the optical Microscope Axioplan II of Zeiss, in which the photomicrographs of this work were taken. The work was developed with support of the CNPq (520901/95-9), PRONEX (0877) FAPERJ (E-26/170.583/95) and CPEG-UFRJ (351905P077-8).

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